JP5462268B2 - Epothilone glycoside compounds and their active ingredients and their applications - Google Patents

Description

  The present invention relates to an epothilone glycoside compound, a preparation method thereof, a drug formulation containing the compound as an active ingredient, and its application to the preparation of drugs for the treatment and prevention of liver cancer, lung cancer and breast cancer.

  Epothilone is a macrolide compound having antitumor activity. For example, the structures of Epothilone A and B, which are commonly seen at present, are as follows.

  Myxobacteria (Sorangium cellulosum) is a single-cell gram-negative gonococcus that is capable of slip migration belonging to the order Myxococcus, Cystobacter, Cystobacteraceae, and Cystobacter, and is present in large amounts in soil. This bacterium can synthesize a wide variety of secondary metabolites, and the diversity of the chemical structure and biological activity mechanism of the metabolites is tremendously equivalent to Streptomyces.

  The main products of epothilone synthesized by myxobacteria are epothilone A and B, but due to their structure, this product is a complex multiene composed of multi-module polyketide synthase and single-module non-ribosomal peptide synthase. It can be expected to be synthesized by the catalytic action of the team complex.

  In 1993, Rechenbach and Hofle et al. Expressed these cytotoxic natural products. In 1995, the Merch Research Laboratories research group found that these compounds have a mechanism of action similar to that of paclitaxel (TAXOL®). Proved. In 1996, Hofle et al. Posted the stereochemical structure of epothilone B and its structural feature was epoxide / thiazole / ketone, so Rechenbach and Hofle et al. Named it “epothilones”. is there.

  Epothilone, similar to paclitaxel (TAXOL®), has the effect of promoting microtubule polymerization and microtubule-stabilizing, and induces a super-stable state by inducing a polymer of microtubule proteins. Inhibiting mitosis and further inhibiting the growth of tumor cells. Epothilone's molecular structure is much simpler than paclitaxel, and has superior water solubility and excellent chemical modification. This product has extremely high activity against tumor cells that are resistant to paclitaxel, is derived from Sorangium cellulosum, and has the power of large-scale fermentation production. Due to the various features described above, epothilone is recognized as a paclitaxel upgrade product and is a new type of anti-tumor drug with a very large market prospect.

  At present, several epothilone analogues have already entered their respective clinical evaluation stages and are being sold on the market. For example, ixabepilone (aza-epothilone B BMS-247550), BMS-310705 (a water-soluble analog of epothilone B), patupilone (epothilone B, EPO906, developed by Novartis), epothilone D (KOS-862, Kosan / Sloan- Kettering / Roche), ZK-EPO and C20-desmethyl-C20-methylsulfanyl-Epo B (ABJ879, Novartis). However, as a result of the search, there were no reports about epothilone glycoside compounds.

  An object of the present invention is to provide an epothilone glycoside compound, a preparation method thereof, a drug formulation containing this compound as an active ingredient, and an application method thereof for preparation of a drug for the treatment and prevention of liver cancer, lung cancer and breast cancer. It is.

  The structure of the epothilone glycoside compound of the present invention is as shown in formula (I) or (II).

その中、

その中、

Among them,

Among them,

  The epothilone glycoside compound is obtained by fermenting a solid and / or liquid of Sorangium cellulosum So0157-2 CCTCC NO: M 208078 and separating the solid or liquid fermentation product. Among them, the above-mentioned myxobacteria (Sorangium cellulosum) So0157-2 CCTCC NO: M 208078 has already been preserved in the Chinese typical culture preservation center (Wuhan University, Wuhan, China) on May 27, 2008.

  The drug formulation used for the prevention and treatment of the liver cancer of the present invention contains a therapeutically effective amount of the above compound and a pharmaceutically acceptable carrier.

  The drug formulation used in the prevention and treatment of lung cancer of the present invention contains a therapeutically effective amount of the above compound and a pharmaceutically acceptable carrier.

  The drug formulation used in the prevention and treatment of breast cancer of the present invention contains a therapeutically effective amount of the above compound and a pharmaceutically acceptable carrier.

  The above-mentioned pharmacologically accepted carrier means an ordinary drug carrier in the pharmaceutical field, such as diluents, excipients such as water, fillers such as starch and saccharose, fibrin derivatives, alginate, gelatin and polyvinylpyrrolidone. Adhesives such as glycerin, active water agents such as glycerin, disintegrants such as agar / calcium carbonate and sodium bicarbonate, absorption accelerators such as quaternary ammonium compounds, surface active agents such as hexadecanol, adsorption of kaolin and bentonite Agents, talc powder, calcium stearate and magnesium, and lubricants such as polyglycol. Moreover, other adjuvants, such as a flavoring agent and a sweetener, can also be put in a formulation.

  The compounds of the present invention are used for the preparation of drugs for the prevention and treatment of liver cancer, lung cancer and breast cancer.

The inventor has revealed that the compound of the present invention has a preventive and therapeutic action on liver cancer, lung cancer, and breast cancer. According to the test, the compound of the present invention has a strong inhibitory effect on human hepatoma cell HepG2 cells at 10 ^-6 M concentration, against human lung cancer A-549 cells and breast cancer MDA-MB-435 cells. Also had a certain degree of inhibitory action. This means that the active site of this compound can be used as a chemical inhibitor for liver cancer, lung cancer and breast cancer, and the compound of the present invention or a drug combination of said compound can be used for the preparation of related drug formulations Explain that.

  The compound of the present invention can be used in patients in need of treatment of the above diseases in the form of a formulation by internal administration, nasal inhalation, rectal or enteral / gastric administration. When used for internal use, it can be used as ordinary solid preparations such as tablets, powders, granules, capsules, etc., and it can be used as liquid preparations such as water or oil suspensions or syrups and elixirs. Can do. In the case of intestinal or gastric administration, it can be used as a solution for injection, water or oil suspension. Preferred forms are tablets, coated tablets, capsules, plugs, nasal sprays and injections, the most suitable being a formulation that can be released to a specific site in the intestine.

  Various dosage forms of the drug formulation of the present invention can be prepared by ordinary production methods in the pharmaceutical field. For example, the active ingredient is mixed with one or several carriers and then made into a dosage form that requires it.

  The preferential active ingredient weight content of the drug formulation of the present invention is 0.1% -99.5%, and the optimum weight content is 0.5-95%.

  The amount of the drug formulation of the present invention varies depending on the administration mode of the drug, the age and weight of the patient, the type and degree of the disease to be treated, and the daily dose is 0.01 to 10 mg / kg body weight However, it is preferentially 0.1 to 5 mg / kg body weight, but can be used once or divided into several times.

  Then, together with the examples, the specialists of the present invention fully understand the present invention, but the present invention is not limited by an arbitrary method.

1． Acquiring strains, protecting them, isolating and purifying epothilone glycoside compounds and testing their antitumor activity (epothilone glycoside A-1 as an example):
The soil sample for collecting bacteria is collected from the shore of the sea lake in Yunnan Province, and the strain is isolated by the following method.

  Place sterile filter paper on a CNST plate, adjust the pH value of the medium to 7.2, add 25 μg / ml concentration of filtered sterilization activion, incubate vertically at 30 ° C, and after 2 days every day under a dissecting microscope In addition to observing the expansion state, the obtained expansion plate of myxobacteria is transferred to a fresh CNST medium and purified. At this time, the pH value of the medium is set to 10.0. Then, after culturing vertically at 30 ° C, the structure of the sub-entity appears after 5 days. The obtained sub-entity is obtained on the already sterilized mycelia of colon gonococci in a WCX plate containing 250 µg / ml kanamycin sulfate. Purification is completed by inoculating and removing the majority of the bacteria and finally inoculating the mucinous bacteria at the edge of the community onto filter paper on CNST (pH 10.0) plates. Scrape off the sub-entities of the already purified, purified myxobacteria on the plate and transfer them to a 1.5 x 3 cm sterile filter paper, then place the filter paper in a sterile strain storage tube and store in a dry place To do. By this process, a starting strain for selection and growth is obtained using the alkali-resistant myxobacteria So0157 capable of producing epothilone as the subsequent strain.

  Using the alkali-resistant myxobacteria So0157 capable of producing the epothilone as a starting strain, the strain is induced and conditioned by repeated solid-liquid continuous intermittent culture.

  The specific method is as follows. First, the strain is cultured in a solid CNST medium at pH 9.0 at 30 ° C, and after 7 days the formation of sub-substances can be observed. Inoculate a 100ml volume of liquid VY / 2 medium, shake culture at 30 ° C, 200rpm for 5 days, then take 10ml of fermentation broth and inoculate 90ml volume of liquid VY / 2 medium, By incubating for 5 days at 30 ° C and 200rpm, taking 10ml of the fermentation broth and inoculating 90ml volume of liquid VY / 2 medium, and incubating for 5 days at 30 ° C and 200rpm. One induction and habituation culture process is completed. The second culture process begins by inoculating 1 ml of the finally cultured fermentation broth into pH 9.0 solid CNST medium and performing reverse culture at 30 ° C. By repeating a plurality of processes by such a method, the obtained strain was fermented in a shaking culture machine, and the target strain was selected by evaluating its growth state and measuring the amount of epothilone produced. In addition, the inventor was able to obtain a strain of the epothilone slime bacterium So0157-2, which has a high yield. This strain has already been identified as a myxobacteria by Shandong University Microbial National Laboratories, and its 16S rDNA sequence information has already been promulgated (DQ256394.1). (Wuhan University, Wuhan, China), and the storage center number is Sorangium cellulosum So0157-2 CCTCC NO: M 208078.

The CNST blending method and blending method used for the isolation, induction and habituation of the above strains are KNO ₃ 0.5 g / L, Na ₂ HPO ₄ 0.25 g / L, MgSO ₄ 7H ₂ O 1 g / L, FeCl ₃ 0.001%. Trace element solution 1 ml / L, agar 1.5%, but adjust to different pH values if necessary. The plate is replaced after sterilization, and after cooling and solidification, aseptic filter paper is applied.

The composition of the WCX medium used for the strain purification is shown in terms of weight percentage, but CaCl ₂ · 2H ₂ O 0.15%, agar 1.6%, and KOH are adjusted to pH 7.0. After sterilization, the amount of 25 μg / ml is put into filtered and sterilized actinidione. Then, the plate is turned upside down, and dense lines are drawn on the surface of the medium, and the virulent Bacilli is used as a food for the formation of submucosal sub-entities. Colon bacilli are prepared in normal LB medium.

The composition of the VY / 2-fold value used for the induction and habituation of the above strain is expressed in terms of a weight percentage, and is active yeast 0.5%; CaCl ₂ 0.08%; VB ₁₂ 0.5 mg / ml; pH 9.0.

Mixing method of the above trace element solutions: MnCl ₂ · 4H ₂ O 0.1 g / L, CoCl ₂ 0.02 g / L, CuSO ₄ 0.01 g / L, Na ₂ MoO ₄ · 2H ₂ O 0.01 g / L, ZnCl ₂ 0.02 g / L, LiCl 0.005g / L, SnCl ₂ · 2H2O 0.005g / L, H ₃ BO ₃ 0.01g / L, KBr 0.02g / L, KI 0.02g / L.

  According to the study of the invention, Sorangium cellulosum So0157-2 CCTCC NO: M 208078 is a bacterium caused by epothilone compounds. The inventor not only detected compounds such as epothilone A / B / C using methods such as solid-state fermentation and continuous measurement of liquid quality and activity tracking, but also epothilone glycoside A having anticancer activity from the extract. -1, A-2, B-1, B-2, C-1 and C-2 could also be separated.

  The inventor uses a liquid quality continuous device to use a glycoside corresponding to epothilone A / B / C (epothilone glycoside A-1, A-2, B-1, B-2, C-1 and C-2). The molecular ion peak of was detected.

1.1 Sorangium cellulosum So0157-2 CCTCC NO: M 208078 Fermentation and Extraction of Compounds By normal culturing method, Sorangium cellulosum So0157-2 CCTCC NO: M 208078 Select and inoculate solid M26 plates. Then, after culturing at 30 ° C for 3-4 days, scraping the cells, inoculating into a shaking culture machine of 50 ml liquid M26 medium, culturing at 30 ° C for 4-5 days, When the growth period is logarithmic, the cells are uniformly mixed with a sterile bottle rotator, inoculated into M26 liquid medium, and expanded. After the expansion culture is finished, the cells are used as a seed for solid fermentation, washed with sterile water, scraped off, and evenly spread on the filter paper of CNST medium. Incubate for 4 days, logarithmic growth, 2% proportionality, spread 1 layer of XAD16 resin on filter paper and continue at 7-9 until fully entering the next metabolic stage at 30 ° C Incubate for days.

CNST formulation and formulation related to the above contents: KNO ₃ 0.5 g / L, Na ₂ HPO ₄ 0.25 g / L, MgSO ₄ · 7H ₂ O 1 g / L, FeCl ₃ 0.001%, trace element solution 1 ml / L, agar 1.5%, pH 7.2. Replace the flat plate after sterilization, and paste a sterile filter paper after cooling and solidification.

Formulation and formulation of the above M26 medium: Potato starch 8g / L, yeast extract 2g / L, peptone 2g / L, glucose 2g / L, MgSO ₄ · 7H ₂ O 1g / L, CaCl ₂ 1g / L, EDTA- FeCl ₃ 1 ml / L, trace element solution 1 ml / L, pH 7.2, solid requires addition of 12 g / L agar powder.

Method of blending the above trace element solutions: MnCl ₂ · 4H ₂ O 0.1 g / L, CoCl ₂ 0.02 g / L, CuSO ₄ 0.01 g / L, Na ₂ MoO ₄ · 2H ₂ O 0.01 g / L, ZnCl ₂ 0.02 g / L, LiCl 0.005g / L, SnCl ₂ · 2H2O 0.005g / L, H ₃ BO ₃ 0.01g / L, KBr 0.02g / L, KI 0.02g / L.

  Collect the solid plate after 7-9 days of culture, scrape M208078 cells and XAD resin on the filter paper with a sterilized scoop, and peel off the filter paper biodegraded by cystobacter with tweezers. , Put both in an oven at 40 ° C to dry the excess water, soak in methanol, filter the soaking liquid with filter paper, and dry at 40 ° C, epothilone glycosides A-1, A Extracts containing -2, B-1, B-2, C-1 and C-2 are obtained.

  By fermenting 10 liters of Sorangium cellulosum So0157-2 CCTCC NO: M 208078 and XAD resin, soaking in methanol, filtering the soaking liquid with filter paper and drying at 40 ° C An extract containing 1.25 g of epothilone glycosides A-1, A-2, B-1, B-2, C-1 and C-2.

1.2 Separation and purification of epothilone glycoside A-1 The fermentation extract of Sorangium cellulosum So0157-2 CCTCC NO: M 208078 was separated by medium pressure liquid phase column chromatography (RP-18, 80 g), Wash off with methanol solution system. From 50% 1500 ml, M1 + M2 (940 mg) is obtained, 65% 700 ml (M3, 100 mg) 75% 700 ml (M4, 250 mg), 300 ml methanol wash column (M5, 82 mg), TLC By developing with inspection, petroleum ether / acetone (3: 2), the flow components M3 and M4 contain spots inlaid by alkaloid color regents. Moreover, there are spots in M3 that are inlaneed by one relatively polar alkaloid color reagent.

M3 is first separated by gel column chromatography, washed out with methanol, and collected automatically, but 3 ml (2600 seconds) is collected in each test tube, TLC inspection is performed, and chloroform / methanol (10 Developed in 1), each fluid component 17-22 (44 mg) and fluid component 23-24 (11 mg) are merged. Fluid component 17-22 is then separated by gel column chromatography, washed out with methanol, and collected automatically, but 3 ml (2600 seconds) is collected in each test tube. Merge 5-8 (35 mg). This fluid component is separated by normal face column chromatography, 0.8 g of silica gel petroleum ether is placed in a column to saturate, and the upper part is dissolved with chloroform. First wash with petroleum ether / acetate ester (10: 1, 41 ml; 5: 1, 48 ml) followed by chloroform / methanol (30: 1). 25 g) is collected, but subsequently separated by normal face column chromatography, placed in a column to saturate 0.6 g silica gel petroleum ether and the upper part is dissolved in chloroform. Chloroform / methanol (40: 1, 41 ml; 35: 1, 36 ml; 30: 1, 31 ml) was washed off by gradient, and about 3-4 ml / tube was collected. -6 (13 mg) and fluid component 7-10 (5 mg) were merged, and the latter was subjected to TLC inspection, but the development of several types of developing agents was one spot. This is explained as a pure compound, the number of which is EPO-E (NMR spectrum measured with CDCl ₃ as solvent).

2． Identification of the structure of the compound EPO-E
The molecular weight of the compound is 625 because the quasimolecular ion peaks of the compound EPO-E obtained from the results of the ESIMS mass spectrum are m / z 626.4 [M + H] ⁺ and 648.3 [M + Na] ⁺ . Moreover, there is a fragment peak m / z 492 with m / z 133 split. According to the results of the high-resolution fast atom bombardment mass spectrum, the molecular formula of this compound is C ₃₁ H ₄₇ NO ₁₀ S (HRFAB ^- MS, actual measured value: m / z 625.7706, calculated value: m / z 625.2921).

C-NMR (including DEPT) of compound EPO-E provides 31 signals, including 6 methyl groups, 7 methylene groups, 12 methine groups and 6 quaternary carbons. include. Δ of ¹ H NMR spectrum 5.21 (s, H-1 ', the anomeric proton), 3.91 (s, H-2'), 4.00 (m, H-3 '), 4.31 (m, H-4') , 3.85 (H-5 ') and 3.78 (dd, H-5') and ¹³ C NMR spectra δ. 108.7 (C-1 '), 79.0 (C-2'), 78.2 (C- The presence of α-D-ribofuranosyl unit was confirmed by signals at 3 ′), 88.1 (C-4 ′) and 66.2 (C-5 ′), and HMQC and HMBC spectrum signals. Further analysis on HMQC and HMBC spectrum-related signals confirmed that the aglycon is epothilone A (data see Table 1). The remote relationship between the proton at C-1 'and C-3 confirmed that the sugar alternative was the C-3 position of epothilone A. Therefore, the compound EPO-E was identified as an epothilone glycoside and named epothilone glycoside A-1, which is a new compound.

^{a 1}H, ¹³C NMR及びHMBC測定データは、室温にて得られ、CDCl_3，は、それぞれ600MHz，150MHz及び600MHzの下で得られる。
^b 特別な説明がない限り、プロトン信号は１Hに帰一化される。

^{a 1} H, ¹³ C NMR and HMBC measurement data are obtained at room temperature, and CDCl ₃ is obtained under 600 MHz, 150 MHz and 600 MHz, respectively.
^b Proton signal is normalized to 1H unless otherwise specified.

3． Compound-Test for Antitumor Activity of Epothilone Glycoside A-1 Screening Method:
Reduction method of methyl-thiazol-tetozolium (MTT) Protein staining method of sulforhodamine B (SRB) Cell lines: HepG2 human liver cancer, A-549 human lung cancer and MDA-MB-435 human Breast cancer Action time: 48-72h
Result evaluation: Invalid: 10 ^-5 mol / L <85%
Weak effect: 10 ^-5 mol / L ≥ 85% or 10 ^-6 mol / L> 50%
Strong effect: 10 ^-6 mol / L> 85% or 10 ^-7 mol / L> 50%
See Table 2 for specific experimental results.

Conclusion: From Table 2, when the concentration of epothilone glycoside A-1 reaches 10 ^-6 M, there is a strong inhibitory effect on hepatoma cell HepG2, human lung cancer cell A-549 and human breast cancer cell MDA- It has a weak inhibitory effect on MB-435. Therefore, since this compound has a selective inhibitory action against tumor cells, the active site of this compound can be a chemical inhibitor of related cancer cells.

調整方法：エポチロングリコシドA-1と乳糖及びトウモロコシ澱粉を混ぜて、水で均一に濡らして、篩にかけてから乾燥し、再び篩にかけてから、ステアリン酸マグネシウムを入れて、均一に錠剤にするが、1枚の重さは240mgで、エポチロングリコシドA-1の含有量は1mgである。

Preparation method: Mix epothilone glycoside A-1, lactose and corn starch, uniformly wet with water, sieve, dry, sieve again, put magnesium stearate into tablets uniformly, The weight of the sheet is 240 mg, and the content of epothilone glycoside A-1 is 1 mg.

調整方法：エポチロングリコシドA-1と乳糖及びステアリン酸マグネシウムを混ぜて、篩にかけ、適当な容器にて均一に混ぜる。得られた混合物はハードゼラチンカプセルに入れるが、1カプセルの重さは200mgで、エポチロングリコシドA-1の含有量は1mgである。

Preparation method: Epothilone glycoside A-1, lactose and magnesium stearate are mixed, sieved, and mixed uniformly in a suitable container. The resulting mixture is placed in hard gelatin capsules, each capsule weighing 200 mg and the content of epothilone glycoside A-1 is 1 mg.

調整方法：エポチロングリコシドA-1と塩化ナトリウムを適当量の注射用水に溶かして、ろ過によって得られた溶液を無菌状態で、アンプル瓶の中に入れる。

Preparation method: Epothilone glycoside A-1 and sodium chloride are dissolved in an appropriate amount of water for injection, and the solution obtained by filtration is put in an ampule bottle under aseptic conditions.

調整方法：エポチロングリコシドB-1と乳糖及びステアリン酸マグネシウムを混ぜて、篩にかけて、適当な容器の中にて均一に混ぜる。得られた混合物はハードゼラチンカプセルに入れるが、1カプセルの重さは200mgで、エポチロングリコシドB-1の含有量は1mgである。

Preparation method: Epothilone glycoside B-1, lactose and magnesium stearate are mixed, sieved and mixed uniformly in a suitable container. The resulting mixture is placed in hard gelatin capsules, each capsule weighing 200 mg and the content of epothilone glycoside B-1 is 1 mg.

調製方法：エポチロングリコシドB-1と塩化ナトリウムを適当量の注射用水に溶かして、ろ過によって得られた溶液を無菌状態で、アンプル瓶の中に入れる。

Preparation method: Epothilone glycoside B-1 and sodium chloride are dissolved in an appropriate amount of water for injection, and the solution obtained by filtration is put in an ampule bottle under aseptic conditions.

調整方法：エポチロングリコシドC-1と乳糖及びトウモロコシ澱粉を混ぜて、水で均一に濡らして、篩にかけてから乾燥し、再び篩にかけてから、ステアリン酸マグネシウムを入れて、均一に錠剤にするが、1枚の重さは240mgで、エポチロングリコシドC-1の含有量は1mgである。

Preparation method: Epothilone glycoside C-1, lactose and corn starch are mixed, wetted uniformly with water, sieved, dried, sieved again, put magnesium stearate into tablets uniformly, The weight of the sheet is 240 mg, and the content of epothilone glycoside C-1 is 1 mg.

調整方法：エポチロングリコシドA-2と乳糖及びステアリン酸マグネシウムを混ぜて、篩にかけ、適当な容器にて均一に混ぜる。得られた混合物はハードゼラチンカプセルに入れるが、1カプセルの重さは200mgで、エポチロングリコシドA-2の含有量は1mgである。

Preparation method: Epothilone glycoside A-2, lactose and magnesium stearate are mixed, sieved, and mixed uniformly in a suitable container. The resulting mixture is placed in hard gelatin capsules, each capsule weighing 200 mg and the content of epothilone glycoside A-2 is 1 mg.

調製方法：エポチロングリコシドB-2と塩化ナトリウムを適当量の注射用水に溶かして、ろ過によって得られた溶液を無菌状態で、アンプル瓶の中に入れる。

Preparation method: Epothilone glycoside B-2 and sodium chloride are dissolved in an appropriate amount of water for injection, and the solution obtained by filtration is placed in an ampule bottle under aseptic conditions.

Claims (1)

A epothilone glycoside compounds shown in formula (I), and is obtained from So0157-2 culture, myxobacteria (Sorangium cellulosum) So0157-2 CCTCC NO: After a solid or liquid fermentation M 208078, the solid or An epothilone glycoside compound obtained by separating a product of liquid fermentation and having antitumor activity.

Here, R ₁ = R ₂ = O, R ₃ = R ₄ = H, R ₅ = glycosyl.